Elucidating the developmental events that lead to the differentiation of a variety of specialized neurons is an important goal for understanding normal nervous system development, for explaining and intervening in congenital birth defects, and for designing neuronal replacement therapies for degenerative CNS diseases. The goals of this research program are to determine the molecules and cellular interactions that regulate the fate choices of embryonic progenitors to contribute to the nervous system. Using an experimental system that allows investigation of maternal molecules and cell-to-cell interactions we will test the function of localized and locally activated molecules that influence the very beginning of an embryonic cell's developmental pathway to becoming neural. To identify the maternal gene products contained in blastomere D1.1 that function in axis specification, including formation of the CNS, cDNA libraries were constructed from translatable RNAs isolated from that blastomere, and screened for pools of mRNAs that induce secondary axial structures. We identified a novel fork head (winged helix) transcription factor and several pools of dorsal axis-inducing cDNAs with this activity.
The specific aims of this proposal are to define both the maternal and zygotic functions of these new genes. The first goal will be accomplished by: a) using animal cap assays to establish where these genes lay in a signaling hierarchy, b) mis-expression and lineage analyses to determine ectopic function, c) polyadenylation assays to establish protein expression regulation and d) maternal gene knock out techniques. The second goal will be accomplish by: a) using animal cap assays to establish which known candidates upregulate or repress the zygotic expression of these new genes, and b) transgenic mis-expression studies. In addition, we identified an interaction between B-tier and C-tier blastomeres, during cleavage stages but prior to the onset of zygotic transcription, that influences whether these cell contribute to the CNS. The signaling system underlying this fate decision will be determined by targeted over-expression of signaling molecules and unique recombinations of signaling blastomeres. These studies will provide fundamental data regarding the earliest maternal regulation and cell-cell interactions that bias embryonic progenitors to give rise to neural tissues. Furthermore they will elucidate the zygotic function of several new genes that are expressed in the neural plat and establish whether these molecules are involved in establishing neuronal identity during early embryogenesis.
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